WHAT IS NPN?

 Non Protein Nitrogen Compounds has been used to monitor the renal function
 The term Non Protein Nitrogen originated in early days of clinical chemistry when
analytic methodology required removal of protein from the sample before
analysis
 NPN includes 15 different substances but there are only 4 important substances n
clinical chemistry:
 UREA
 CREATININE
 URIC ACID
 AMMONIA
UREA
Highest NPN concentration in the blood
Major excretory product of protein catabolism
Formed in the liver from amino groups (-NH2) and free
ammonia generated during protein catabolism
BLOOD UREA NITROGEN ( BUN) is the term used to refer
urea determination
UREA NITROGEN ( UN) is a more appropriate term
PHYSIOLOGY
Protein metabolism produces amino acids that can be
oxidized to produce energy or stored as fat and glycogen
RELEASES NITROGEN, which is converted to:
UREA and excreted as a waste product
 Most of the urea in the glomerular filtrate is excreted in
the urine
<10% of urea are excreted through the gastrointestinal
tract and skin.
CLINICAL APPLICATION

Measurement of urea is used to evaluate the renal

function, to assess hydration status, to determine nitrogen
balance, to aid in the diagnosis of renal disease, and to
verify adequacy of dialysis.
Assays for urea were originally performed protein-free
filtrate of whole blood and based on measuring the
amount of nitrogen in a sample
UREA NITROGEN

Specimen requirement:

1. SERUM
2. URINE
BLOOD UREA NITROGEN (BUN TEST)
BLOOD UREA NITROGEN
(BUN), SERUM
BLOOD UREA NITROGEN (BUN), SERUM
Specimen: SERUM
Patient Preparation: Fasting
Collection Container/Tube:
Preferred: Serum gel
Acceptable: Red top
Submission Container/Tube: Plastic vial
Specimen Volume: 0.5 mL
Collection Instructions:
1. Serum gel tubes should be centrifuged within 2 hours of collection.
2. Red-top tubes should be centrifuged and aliquoted within 2 hours of collection.
Specimen Type:
Serum
Temperature:
Refrigerated (preferred)
Time:
7 Days
• Males
• 1-17 years: 7-20 mg/dL
• > or =18 years: 8-24 mg/dL
• Reference values have not been established for
patients who are <12 months of age.

• Females
• 1-17 years: 7-20 mg/dL
• > or =18 years: 6-21 mg/dL
• Reference values have not been established for
patients who are <12 months of age.
UREA NITROGEN 24 HOURS TEST
 UREA NITROGEN 24 HOURS TEST
Specimen type: 24-hour urine

Preferred collection container: 24-hour urine container (no

preservative)
Specimen required: 5 mL aliquot of urine; minimum 1 mL.

Special notes: Preferred specimen container is the 24-

hour urine container with NO
preservative. Mix well. Estimate total
collection volume from 24-hour urine
container. Remove required aliquot.
Record total volume on both aliquot
tube and request slip (if applicable).

Patient preparation: Patient should not be taking excessive

amounts of vitamin C as vitamin C will
interfere with test.
Reference Interval

UREA NITROGEN
ADULT
Plasma or 6–20 mg/dL (2.1–7.1 mmol
serum urea/day)
Urine, 24-h 12–20 g/day (0.43–0.71 mol
urea/day)
Pathophysiology
AZOTEMIA
ELEVATED CONCENTRATION OF UREA
IN THE BLOOD
UREMIA OR UREMIC SYNDROME
 VERY HIGH PLASMA UREA CONCENTRATION ACCOMPANIED BY RENAL FAILURE
 THIS CONDITION IS EVENTUALLY FETAL IF NOT TREATED BY DIALYSIS OR TRANSPLANTATION
 CONDITIONS CAUSING INCREASED PLASMA UREA ARE CLASSIFIED ACCORDING TO CAUSE
INTO THREE MAIN CATEGORIES:
 PRERENAL
 RENAL
 POSTRENAL
PRERENAL AZOTEMIA

 CAUSED BY REDUCED RENAL BLOOD FLOW.

 LESS BLOOD IS DELIVERED THROUGH KIDNEY;
 CONSEQUENTLY LESS UREA IS FILTER.
 CAUSATIVE FACTORS:
 CONGESTIVE HEART FAILURE
 SHOCK
 HEMMORRHAGE
 DEHYDRATION
 OTHER FACTOR RESULTING IN A SIGNIFICANT DECREASE IN BLOOD VOLUME
 DECREASE RENAL FUNCTION CAUSES:
 INCREASE IN PLASMA UREA CONCENTRATION
 AS A RESULT, COMPROMISED UREA EXCRETION
RENAL

 CAUSES OF ELEVATED UREA INCLUDE ACCUTE AND CHRONIC RENAL FAILURE;

 GLOMERULAR NEPHRITIS
 TUBULAR NECROSIS
 OTHER INTRINSIC RENAL DISEASE
POST RENAL AZOTEMIA

 CAN BE DUE TO OBSTRUCTION OF URINE FLOW ANYWHERE IN THE URINARY TRACT BY RENAL
CALCULI;
 TUMORS OF THE BLADDER OR PROSTATE OR SEVERE INFECTION
 MAJOR CAUSES OF DECREASE
PLASMA CONCENTRATION

 LOW PROTEIN INTAKE

 SEVERE LIVER DISEASE
 DURING LATE PREGNANCY AND INFANCY AS A RESULT OF INCREASE PROTEIN SYNTHESIS
Blood Uric Acid
Uric Acid

 Major product of PURINE metabolism

 It is the final breakdown of Nucleic Acids catabolism in humans
 Formed by Xanthine by the action of Xanthine Oxidase in the liver and intestine
 Freely filtered, partially reabsorbed and secreted in the renal tubule
 Weak Acid; at pH 7.4, >95% exists as Monosodium Urate (MSU)
 1g of uric acid is excreted normally.
Uric Acid derived from 3 sources:
Catabolism of ingested
nucleoproteins;
Catabolism of endogenous
nucleoproteins;
Direct transformation of
endogenous purine nucleotides.
Reference Values: (Uricase)

Male: 3.5 - 7.2 mg/dL (0.21 - 0.43

mmol/L)
Female: 2.6 - 6.0 mg/dL (0.16 – 0.36
mmol/L)
Disease Correlation

1.Hyperuricemia
2.Hypouricemia
Hyperuricemia

1. GOUT
 A disease found primarily in males and first diagnosed between 3rd and 5th decade of life.
 Pain and inflammation of the joints (Acute inflammatory Arthritis)
 Presence of “birefringent crystals in the synovial fluid”
 Highly susceptible to nephrolithiasis.
Hyperuricemia

2. INCREASED NUCLEAR METABOLISM

 Seen in leukemia, lymphoma, multiple myeloma or polycythemia, hemolytic and
megaloblastic anemias.
 Monitoring is important to avoid nephrotoxicity
 Allopurinol = used for treatment
Hyperuricemia

3. CHRONIC RENAL DISEASE

 due to the decreased GFR and tubular secretion
 > 10 mg/dL levels of plasma uric acid cause urinary tract calculi
Hyperuricemia

4. Lesch – Nyhan Syndrome (Inborn errors of Purine Metabolism)

 Defeciency of hypoxanthine-guanine phosphoribosyl transferase (HGRPT)
Other causes of HYPERURICEMIA:

1. Secondary to glycogen storage disease

2. Toxemia of pregnancy and lactic acidosis
3. Increased dietary intake
4. Ethanol consumption
Hypouricemia

1. Fanconi’s syndrome – renal-type aminoaciduria

2. Wilson’s disease
3. Hodgkin’s disease
Methods

 Fasting may not be required, but for diagnosis purposes, fasting sample is preferred.
 Uric Acid is stable in both serum and urine for 3 days at room temperature.
 Potassium oxalate (anticoagulant) should not be used.
 Ascorbic acid and bilirubin are the major interferences.
Chemical Methods

Principle: Reduction – oxidation (Redox) Reaction

NaCN / NaCO3

Uric acid + Phosphotungstic Acid Tungsten blue + Allantonin +

CO2
A. Sodium cyanine (NaCN) = Folin Brown
Newton Benedict
B. Sodium carbonate (Na2CO3) = Archibald Caraway
Henry
Logphase: the incubation period after the addition of an alkali (NaCN/Na2CO3) to inactivate
non-uric reactants
 Enzymatic Methods

URICASE METHOD
 Most simpliest and specific method
 Uric acid has a UV absorbance peak at 293nm
 Allatonin does not have a UV peak at that wavelength
Principle: the enzyme uricase oxidizes uric acid to form allatonin. Uric acid has a max. peak of
absorption of 293nm. The resultant product (allatonin) has no absorption at this wavelength. The
decrease in the absorbance is proportional to the concentration of uric acid present in the sample.
uricase
Uric Acid + O2 allatonin + CO2 + H2O
Isotope Dilution Mass Spectrometry (ID-
MS)

Reference Method
CREATININE
Desoyo, Dominic
Divino, Jean Queenbell
Maturan, Gia Mae
What is creatinine?

 is a waste product produced by muscles from the breakdown of a compound called

creatine and creatine phosphate.
 It is removed from the body by the kidneys
 Why get tested?
 To evaluate the health of your
kidneys.
 To help diagnose kidney disease.
 To monitor treatment for kidney
disease.
When to get tested?
 Routinely as part of a
comprehensive or basic
metabolic panel when you
have signs and symptoms
that may be due to kidney
disease or damage.
10 c0mmon
habits that
damage
kidney
1. Not emptying your bladder early.
2. Not drinking enough water.
3. Taking too much salt.
4. Not treating common infections quickly and
properly.
5. Eating too much meat.
6. Not eating enough.
7. Painkiller abuse
8. Missing your drugs
9. DRINKING TOO MUCH ALCOHOL!!!!!
10. Not resting enough.
What are the sample required?

 Serum
– Plastic Tubes
- With or without gel barrier
- Glass Tubes were not tested
 Plasma
– Plastic Tubes
- Lithium Heparin/Sodium Heparin
- EDTA is not recommended
 Urine
- Collection at <24h with no
preservatives
- Inspect for particulates
Creatinine in urine and plasma

 Normal serum creatinine level is 0.7 to 1.4 mg/dl and serum creatinine level is 0.2 to 0.4
mg/dl.
 The amount of creatinine excreted is proportional to the total creatinine phosphate
content of the body.
- Therefore can be used to estimate muscle mass.
 Serum creatinine is a sensitive indicator of kidney disease (Kidney Function Test)
- Because normally creatinine is rapidly removed from the blood and excreted.
 The amount of creatinine in urine is used as an indicator for the proper collection of 24hrs
urine sample (Normal Urinary Output is 15-25 mg/kg/dl).
The diagnostic function of creatinine

If the kidneys are damaged or impaired and cannot work normally

The amount of creatinine in urine goes down while its level in blood
goes up

Creatinine has been found to be a fairly reliable indicator of kidney

function
SUMMARY OF ANALYTIC METHODS ----- CREATININE
CHEMICAL METHODS BASED ON JAFFE REACTION

Jaffe Reaction In alkaline solution, creatinine

+picrate -> red orange complex

Jaffe Kinetic Jaffe reaction performed directly
on sample; detection of color
formation timed to avoid
interference or non-creatinine
chromogens
Positive bias from -keto acids and
cephalosporins; requires
automated equipment for
precision

Jaffe with adsorbent Creatinine in protein-free filtrate Adsorbent improves specificity;

adsorbed onto Fuller’s earth, then previously considered reference
reacted with alkaline picrate to method
form colored complex

Jaffe without adsorbent Creatinine in protein-free filtrate Positive bias from ascorbic
reacts with alkaline picrate to form acid,glucose, glutathione, -keto
colored complex acids, uric acid and
cephalosporins
ENZYMATIC METHOD

In a series of enzymatically Adapted for use as dry slide

Creatininase – H2O2 catalyzed reactions, creatinine is method; potential to replace
hydrolyzed to creatine,which is Jaffe; no interference from
converted to sarcosine and urea. acetoacetate or cephalosporins;
Sarcosine is oxidized to glycine, some positive bias due to
CH2O and H2O2. Peroxidase- lidocaine
catalyzed oxidation of a colorless
substrate produces a colored
product + H2O2
In a series of reactions catalyzed Lacks sensitivity; not widely used
Creatininase - CK by the enzymes creatininase,
creatine kinase, pyruvate kinase
and lactate dehydrogenase,
NAD+ is produced and measured
as a decrease in absorbance

OTHER METHODS
Detection of characteristic Highly specific; accepted
Isotope dilution mass spectrometry fragments following ionization; reference method
quantification using isotopically
Sources of error
Ascorbate, glucose, -keto acids
and uric acid may increase Patients taking cephalosporin
creatinine concentration measured antibiotics may have falsely elevated
by Jaffe Reaction esp. at results when Jaffe Reaction is used.
temperatures above 30C.

Dopamine is known to affect both Lidocaine causes a positive bias in

enzymatic and Jaffe methods. some enzymatic method.
Creatine kinase

 Is responsible for the generation

of energy in contractile muscular
tissues.
 CK levels are changed in
disorders of cardiac and skeletal  Serum total CK is increased in:
muscle. - Crush injuries (Damage of Skeletal Muscles)
 Is required for conversion of - Myocardial Infarction (Damage of Heart
creatinine into creatine Muscle)
phosphate.
 Has 3 isoenzymes:
CK-MM Mainly in Skeletal
Muscle
CK-MB Mainly in Heart
Muscle
CK-BB Mainly in Brain
REFERENCE INTERVALS OF CREATININE
Adult (Plasma/Serum) Jaffe Method Enzymatic Method

Male 0.9-1.3 mg/dL 0.6-1.1 mg/dL

(80-115 mol/L) (53-97 mol/L)

Female 0.6-1.1 mg/dL 0.5-0.8 mg/dL

(53-97 mol/L) (44-71 mol/L)

Child 0.3-0.7 mg/dL 0.0-0.6 mg/dL

(27-62 mol/L) (0-53 mol/L)

Adult Urine
24h

Male 800-2000 mg/dL

(7.1-17.7 mmol/d)

Female 600-1,800 mg/d

(5.3-15.9 mmol/d)
pathophysiology
Creatinine
 Elevated creatinine concentration is
associated with abnormal renal function
esp. as it relates to glomerular function.
Plasma concentration of creatinine is
inversely proportional to the clearance of
creatinine. Therefore when plasma
creatinine concentration is elevated GFR
is decreased indicating renal damage.
Plasma creatinine is a relatively insensitive
marker and may not be measurably
increased until renal function has
deteriorated more than 50%.
Creatine
 In muscle disease such as muscular
dystrophy, poliomyelitis, hyperthyroidism
and trauma, both plasma creatine and
urinary creatinine are often elevated.
Plasma concentrations are usually normal
in these patients. Measurement of
creatine kinase is used typically for the
diagnosis of muscle disease because
analytic methods for creatine are not
readily available in most clinical
laboratories. Plasma concentrations is not
elevated in renal disease.
AMMONIA
INTRODUCTION

Ammonia is formed in the deamination of amino

acid.

It is removed from the circulation and converted

to urea in the liver .
Biochemistry

Ammonia is produced in the catabolism of amino

acids and by bacterial metabolism in the lumen of
the intestine .

Some endogenous ammonia results anaerobic ,

metabolic reactions that occur in the skeletal
muscle during exercise .
 free ammonia is toxic however , ammonia is present in the plasma in
low concentrations .

 Ammonia is consumed by the parenchymal cells of the liver in the

production of urea.

 At normal physiologic pH , most ammonia in the blood exists as

ammonium ion (NH4+)

 Ammonia is excreted as ammonium ion by the kidney and acts to

buffer urine .
URE
A
Clinical applications

Clinical condition in which blood ammonia

concentrations provides useful information are hepatic
failure.
Severe liver disease is the most common cause of
disturbed ammonia metabolism .
The monitoring of blood ammonia maybe used to
determine prognosis , although correlation between the
extent of hepatic encephalopathy and plasma
ammonia concentration is not always consistent .
 Arterial ammonia concentration is a better indicator of the
severity of disease.

 Reye’s syndrome occurring most commonly in children , is a

serious disease that can be fatal.

 It is preceded by a viral infection and the administration of

aspirin

 Reye’s syndrome is an acute metabolic disorder of the liver

and autopsy findings show severe fatty infiltration of the organ.
Survival reaches 100% if plasma NH3 concentration
remains below 5 times normal.

Ammonia is used in the diagnosis of inherited

deficiency of urea cycle enzymes.

Assay of blood ammonia can be used to monitor

hyperlimentation therapy and the measurement of
urine ammonia can be used to confirm the ability of
the kidneys to produce ammonia
REYE’S SYNDROME
Methods

 Two approaches have been used for the measurement of plasma

ammonia

 One is the 2 STEP APPROACH in which ammonia is isolated from the

sample and then assayed .

 The second involves DIRECT MEASUREMENT of ammonia by an

enzymatic method or ion-selective electrode . Assays detect NH3 or
NH4+.
 One of the 1st analytic methods for ammonia developed by
Conway in 1935, exploited the volatility of ammonia to separate
the compound in the micro-diffusion chamber
 Ammonia can be measured by an enzymatic method using
glutamate dehydrogenase.

 The decrease in absorbance (ADP)- ( Adenosine 5’

diphosphate) is added to the reaction mixture increase the
rate of the reaction and to stabilize GLDH (Glutamate
dehydrogenase). The method is used on many automated
systems and is available as a prepared kit from numerous
manufacturers
 Specimens requirements and
interfering substances

Careful specimen handling is extremely important for

plasma ammonia assays.

Whole blood ammonia concentration increases rapidly

following specimen collection because of in vitro amino
acid deamination.
Venous blood should be obtained without trauma and
placed on ice immediately.
Heparin and EDTA are suitable anticoagulants.

Commercial collection containers should be

evaluated for ammonia interference before a
new lot is put into use.

Samples should be centrifuged at 0-4 degrees

celcius within 20 minutes
Frozen plasma is stable for several days at -20
degrees celcius
Sources of Error

 Ammonia contamination is a potential problem in the laboratory

measurement of ammonia . Precautions must be taken to minimize
contamination in the laboratory in which the assay is performed.

 Elimination of sources of ammonia contamination can significantly

improve the accuracy of ammonia assay results
Sources Of Contamination Include
tobacco smoke
urine
and ammonia in detergents , glassware , reagents and water

the ammonia content of serum-based control material is unstable

. Frozen aliquots of human serum albumin containing known
amounts of ammonium chloride , or ammonium sulfate maybe
used .
Solution containing known amounts of ammonium sulfate are
commercially available
Reference interval

 Values obtained vary somewhat with the method used . Higher concentration are seen in
newborns.
 Pathophysiology

 In severe liver disease in which there is significant collateral circulation

or if parenchymal liver cell function is severely impaired ; ammonia is
not removed from the circulation and blood concentration increases.

 High concentration of NH3 are neurotoxic and often associated with

encephalopathy .
 Toxicity may be partly a result of extracellular glutamate
concentration and subsequent of adenosine triphosphate in the
brain.

 Hyperammonemia is associated with inherited deficiency of urea

cycle enzymes.

 Measurement of plasma ammonia is important in the diagnosis

and monitoring these inherited metabolic disorders